High-Affinity N-(2-Hydroxypropyl)methacrylamide Copolymers with Tailored N-Acetyllactosamine Presentation Discriminate between Galectins

Article abstract of DOI:10.1021/acs.biomac.9b01370

N-Acetyllactosamine (LacNAc; Galβ4GlcNAc) is a typical disaccharide ligand of galectins. The most abundant members of these human lectins, galectin-1 (Gal-1) and galectin-3 (Gal-3), participate in a number of pathologies including cancerogenesis and metas

Full text of DOI:10.1021/acs.biomac.9b01370

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Article
High-affinity HPMA copolymers with tailored N-
acetyllactosamine presentation discriminate between galectins
Marina Rodrigues Tavares, Markéta Bláhová, Lieselotte Sedláková, Lothar Elling, Helena
Pelantová, Rafal Konefal, Tomáš Etrych, Vladimir Kren, Pavla Bojarova, and Petr Chytil
Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.9b01370 • Publication Date (Web): 06 Jan 2020
Downloaded from pubs.acs.org on January 8, 2020
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High-affinity HPMA copolymers with tailored N-
acetyllactosamine presentation discriminate between
galectins
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Marina Rodrigues Tavares,a Markéta Bláhová,a Lieselotte Sedláková,b,c Lothar Elling,d
Helena Pelantová,b Rafal Konefał,a Tomáš Etrych,a Vladimír Křen,b Pavla Bojarová,*b,c
and Petr Chytil*a
a
Institute of Macromolecular Chemistry, Czech Academy of Sciences, Heyrovského
náměstí 2, CZ-162 06 Prague 6, Czech Republic. E-mail: chytil@imc.cas.cz
b
Institute of Microbiology, Czech Academy of Sciences, Vídeňská 1083, CZ-142 20,
Prague 4, Czech Republic. E-mail: bojarova@biomed.cas.cz
c
Department of Health Care Disciplines and Population Protection, Faculty of
Biomedical Engineering, Czech Technical University in Prague, Sítná sq. 3105, CZ-272
01 Kladno, Czech Republic.
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dInstitute of Biotechnology and Helmholtz Institute for Biomedical Engineering, RWTH
Aachen, Pauwelstr. 20, D-52079 Aachen, Germany.
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ABSTRACT
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N-Acetyllactosamine (LacNAc; Galβ4GlcNAc) is a typical disaccharide ligand of
galectins. The most abundant members of these human lectins, galectin-1 (Gal-1) and
galectin-3 (Gal-3), participate in a number of pathologies including cancerogenesis and
metastatic formation. In this study we synthesized a series of fifteen N-(2-
hydroxypropyl) methacrylamide (HPMA) based glycopolymers with varying LacNAc
amount and presentation, and evaluated the impact of their architecture on the binding
affinity to Gal-1 and Gal-3. The controlled radical RAFT (reversible addition-
fragmentation chain transfer) copolymerization technique afforded linear polymer
precursors with comparable molecular weights (Mn ≈ 22,000 g mol−1) and a narrow
dispersity (Đ ≈ 1.1). The precursors were conjugated with the functionalized LacNAc
disaccharide (4-22 mol. % content in glycopolymer), prepared by enzymatic synthesis
under the catalysis by β-galactosidase from Bacillus circulans. The structure-affinity
relationship study based on ELISA-type assay revealed that the type of LacNAc
presentation, individual or clustered on bi- or trivalent linkers, brings a clear
discrimination (almost 300-fold) between Gal-1 over Gal-3, reaching avidity to Gal-1 in
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nanomolar range. While Gal-1 strongly preferred a dense presentation of individually
distributed LacNAc epitopes, Gal-3 preferred clustered LacNAc presentation. Such a
strong galectin preference based just on the structure of a multivalent glycopolymer type
is exceptional. The prepared non-toxic, non-immunogenic and biocompatible
glycopolymers are prospective for therapeutic applications requiring selectivity for one
particular galectin.
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KEYWORDS
HPMA copolymer; galectin; glycopolymer; N-acetyllactosamine; selectivity
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INTRODUCTION
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Current research devoted to cancer treatment has been facing challenges to overcome the
limitations of conventional anti-cancer therapeutics including the lack of selectivity,
toxicity, and the resistance of tumor cells. Therefore, the development of biocompatible
nanocarrier systems selectively targeting tumor tissues triggers an increasing interest
aiming to improve the efficacy and safety of anti-cancer therapy.1,2 Moreover, the
therapeutic potential of these systems may further be improved by attaching specific
targeting moieties2–4 such as antibodies,5 oligopeptides,6,7 or carbohydrates.8
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Polymer carriers based on N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers
have been receiving considerable attention for their ability to influence the
pharmacokinetics of various bioactive compounds, prolong their circulation time in vivo
and deliver them to their biological targets.2,4,9 Their potential as carbohydrate carriers
targeting lectins was shown previously.10 Among other favorable properties, HPMA
copolymers are biocompatible, non-immunogenic, non-toxic and water-soluble
biomaterials. The powerful technique of the controlled radical RAFT (reversible addition-
fragmentation chain transfer) polymerization enables the synthesis of well-defined
polymers carrying suitable functions for attaching bioactive molecules.2,4,9,11 These
polymers have adjustable molar mass, narrow molar mass distribution and high end-group
fidelity – important characteristics for the polymer conjugates designed for medical
purposes.11
Galectins are glycan-binding soluble proteins overexpressed in many types of cancer both
inside the cancer cells and extracellularly.12 They are involved in tumor cell adhesion,
metastasis,13 tumor angiogenesis,14 proliferation and growth of cancer cells15 as well as in
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their escape from the host immune system.16 Alterations in the galectin expression in
human cells have been linked to the development of cancer. Therefore, galectins are
considered a promising target for the anti-cancer therapy and their presence in the patient
blood can indicate the prognosis of the ongoing disease,12,17–19 thus meeting basic
requirements for a cancer biomarker.12,18–20 There are twelve human galectins known.21
However, most studies focus on the two most common and best known representatives –
prototype-type galectin-1 (Gal-1)22 and chimera-type galectin-3 (Gal-3).23
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The carbohydrate binding domains of both Gal-1 and Gal-3 contain a complex diversified
binding groove for carbohydrate ligands based on the β-galactoside moiety. Gal-3
naturally recognizes poly-LacNAc, i.e. [β3Galβ4GlcNAc]n structure, in contrast to Gal-1,
which only binds the terminal disaccharide motif of LacNAc type. Due to the similar
binding preferences of these two most abundant galectins, it is very difficult to prepare
selective inhibitors acting on one of them only. In the case of chimera-type Gal-3, the
multivalency effect evoked by interaction with multivalent disaccharide ligands probably
occurs primarily via the statistical rebinding mechanism, and, therefore, it results in rather
low avidities.24 Avidity is a result of cumulative affinities of individual glycans on the
joint carrier. In contrast, in lectins with several binding sites such as Gal-1, the
multivalency effect12,19,25 may often reach up to 106-fold as a result of the chelation
mechanism.12 However, the molecular fundaments of galectin function are not fully
understood yet and novel information and questions still arise, suggesting a highly
complex nature of these processes.26
Selective galectin targeting, based on a known molecular mechanism, helps to better
intercept and decode the underlying biological processes in their complexity. Moreover,
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robust selectivity, in combination with high binding affinity, enables to exercise efficient
targeted galectin inhibition in the presence of less specific cell surface glycans and
abundant serum glycoproteins, which is a prerequisite for a successful translation into
future clinical practice. The variety of published multivalent systems for galectin
inhibition, which are based on readily available natural ligands such as lactose
(Galβ4Glc) or LacNAc (Galβ4GlcNAc), exhibit no considerable selectivity between
galectins.12 Selectivity can only be accomplished by appropriate choice of the glycan
epitope. For example, TF antigen (Galβ3GalNAc),12 LacdiNAc (GalNAcβ4GlcNAc)
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disaccharide8,27–29
or
the
complex
tetrasaccharide
of
GalNAcβ4GlcNAcβ3Galβ4GlcNAc12,18,19,25 are reported to be selective for Gal-3 in
contrast to Gal-1. A high selectivity has also been demonstrated in small-molecule
inhibitors of galectins, especially those based on the aryl-substituted galactose
scaffold.30,31 These compounds, however, pose a considerable synthetic challenge, which
is also reflected on the overall synthetic yields. Very recently, impact of glycan density on
graft polymers on the selectivity between galectins has been shown by Zhou et al.32 To
our knowledge, no macromolecular ligands selective for Gal-1 have been reported up to
date.
LacNAc is a common disaccharide ligand for both Gal-1 and Gal-3.12 We present here a
novel synthetic approach using LacNAc disaccharide that affords HPMA-based
glycopolymers acting as selective high-affinity ligands of galectins. Their avidity to Gal-1
and Gal-3 was tuned not only by the amount of LacNAc epitope on the polymer carrier
(further termed as “epitope density”) but also by the structure of the linker that presents
LacNAc either individually or in bi- or trivalent clusters. As a result, the best
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glycopolymers reached avidities to Gal-1 in nanomolar range. It is an outstanding
example of a multivalent biocompatible carrier that can reach selectivity between these
two galectins of over two orders of magnitude, particularly, using a disaccharide ligand as
simple as LacNAc. The straightforward synthesis and properties of the present
glycopolymers predestine them for biomedical applications, possibly leading from the
bench to the bedside.
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EXPERIMENTAL SECTION
Materials
The preparation of the β-galactosidase from Bacillus circulans was from Daiwa Kasei,
Ltd. (Shiga, Japan). 2,2′-Azobisisobutyronitrile (AIBN), 2-cyanopropan-2-yl
dithiobenzoate (CTA), 2-thiazoline-2-thiol, 4-(dimethylamino)pyridine (DMAP), -
alanine, CuBr, dimethyl sulfoxide (DMSO), methacryloyl chloride, methanol, methyl 3,5-
dihydroxybenzoate
(8),
methyl-3,4,5-trihydroxybenzoate
(9),
N-(3-
dimethylaminopropyl)-N′-ethylcarbodiimide
hydrochloride
(EDC.HCl),
N-Boc-
ethylenediamine, N,N-diisopropylethylamine (DIPEA), N,N-dimethylacetamide (DMA),
N,N-dimethylformamide (DMF), and t-butanol were purchased from Sigma-Aldrich
(Czech Republic). 1-Aminopropan-2-ol was from TCI Europe (Belgium) and propargyl
bromide from Acros Organics (Czech Republic). Benzotriazole-1-yl-oxy-tris-pyrrolidino-
phosphonium hexafluorophosphate (PyBOP) and trifluoroacetic acid (TFA) were from
Iris Biotech (Germany). The solvents for NMR were from the following producers: D2O
(99.96 atom % D) from VWR Chemicals (Leuven, Belgium), and CD3OD (99.80 atom %
D) from VWR Chemicals (Leuven, Belgium). LacNAc (7; Galβ4GlcNAc; β-D-
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galactopyranosyl-(14)-2-acetamido-2-deoxy-D-glucopyranose) was from Carbosynth
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(UK). All other solvents and chemicals were of analytical grade.
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Characterization Methods
Mass Spectrometry (MS)
Mass spectra of compounds 4-6 and 14-17 were acquired with an LTQ Orbitrap XL
hybrid mass spectrometer (Thermo Fisher Scientific, Waltham, USA) using an
electrospray ion source and injection through a 2 μL loop (4-6) or 5 μL loop (14-17).
Methanol/water (4:1 v/v) was used as a mobile phase at a flow rate of 30 μL min−1 (4-6)
or 100 μL min−1 (14-17). Internal calibration of the mass in the negative mode was
performed with a lock mass of deprotonated palmitic acid. For the negative ion mode,
spray voltage, capillary voltage, tube lens voltage, and capillary temperature were 5.0 kV,
-25 V, -125 V, and 275 °C, respectively. For the positive ion mode, spray voltage,
capillary voltage, tube lens voltage, and capillary temperature were 5.0 kV, 9 V, 150 V,
and 275 °C, respectively. The Orbitrap mass spectra were recorded at the resolution of
100,000.
Nuclear Magnetic Resonance (NMR) Spectroscopy
NMR spectra of carbohydrates and glycosylated branched linkers were measured by
Bruker Avance III 600 MHz spectrometer (1H: 600.23 MHz; C: 150.93 MHz for
13
compounds 4, 5, 16, and 17) or by Bruker AVANCE III 700 MHz spectrometer (1H:
700.13 MHz; 13C: 176.05 MHz for compound 6) in D2O (for compounds 4, 5, 6, and 17)
and CD3OD (for compound 16) at 303 K (compounds 4, 6, 16, and 17) or 298 K
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(compound 5). H NMR, C NMR, gCOSY, gHSQC, gHMBC, and 1D-TOCSY spectra
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were obtained using standard manufacturer’s software.
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1H NMR spectra of polymer precursors and glycopolymers were acquired with Bruker
Avance III 600 (or Bruker Avance DPX 300) spectrometer operating at 600.2ꢀMHz (or
300.1 MHz). The NMR spectra of all samples were measured in 5 mm NMR tubes using
CDCl3, CD3OD, DMSO-d6 or D2O. Typical conditions for measurements of the spectra
were as follows: π/2 pulse width 18 µs (15.6 µs), relaxation delay 15 s (10 s), spectral
width 12 kHz (6.6 kHz), acquisition time 2.73 s (4.95 s), 16-200 scans. The Bruker
Avance DPX 300 MHz spectrometer was used for recording spectra of compounds 10
and 11 (CDCl3) and compound 12 (CD3OD). Bruker Avance III 600 MHz spectrometer
was applied for measurements of compounds 13-15 and 20-24 (DMSO-d6) as well as
compounds 18-19 and 25-39 (D2O).
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High Performance Liquid Chromatography (HPLC)
The composition of transglycosylation reaction mixtures as well as the purity of prepared
carbohydrates was analyzed by hydrophilic interaction liquid chromatography (HILIC)
using Shimadzu Prominence LC analytical system. It was composed of the binary HPLC
pump Shimadzu LC-20AD, cooling autosampler Shimadzu SIL-20ACHT, the column
oven Shimadzu CTO-10AS column oven, the system controller Shimadzu CBM-20A, and
the diode array detector Shimadzu SPD-M20A (Shimadzu, Japan). Samples (1 µL)
dissolved in acetonitrile/water (3:1, v/v) were run through the HILIC column TSKgel
Amide80 5 µm (250 × 4.6 mm, Tosoh Bioscience, Germany) at a flow rate of 1 mL min−1
at 27 °C. For elution, binary gradient was employed: 22% B for 0-7 min, 22-35% B for 7-
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20 min; 35% B for 20-25 min; and 35-22% B for 25-26 min where A is 100% acetonitrile
and B is water. The detection was performed at 200 nm. The purity of the monomers
HPMA (20) and N-methacryloyl-β-alanine thiazolidine-2-thione (MA-AP-TT; 21) for
polymer synthesis and the course of reactions to obtain compounds 16-19 and 25-39 were
monitored on a Shimadzu HPLC system equipped with a C18 reversed-phase Chromolith
Performance RP-18e column (4.6 × 100 mm, Merck Millipore) and a diode array detector
(Shimadzu SPD-M20A), in a mobile phase of water/acetonitrile/0.1% TFA with a
gradient of 5-95% v/v acetonitrile at a flow rate of 5 mL min−1.
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Size Exclusion Chromatography (SEC)
The number-average molecular weight (Mn), weight-average molecular weight (Mw) and
dispersity (Ð) of polymer precursors 22-24 and glycopolymers 25-39 were determined by
a Shimadzu HPLC system equipped with size exclusion chromatography (SEC) columns
(TSKgel Super SW3000, 4.6 × 300 mm, 4 µm, for polymers 22-27, 30-32 and 35-37;
Superose 6, 10 x 300 mm for glycopolymers 28, 29, 33, 34, 38 and 39). Optilab-rEX
differential refractometer index (RI) detector, multi-angle light scattering (MALS)
detector (DAWN HELEOS II, Wyatt Technology Co., USA) and SPD-M20A photodiode
array detector (Shimadzu, Japan) were used. methanol/0.3 M sodium acetate buffer, pH
6.5 (4/1, v/v) at a flow rate of 0.3 mL min−1 was used as a mobile phase for the TSKgel
column, and 0.3 M sodium acetate buffer, pH 6.5 at a flow rate of 0.5 mL min−1 was used
for the Superose column. The Mw and Ð were calculated using ASTRA VI software. The
Optilab®-rEX detector enabled direct determination of refractive increment (dn/dc) of the
polymers, and the solvent refractive index provided 100 % recovery of the injected
sample from the column.
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Dynamic Light Scattering (DLS)
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The hydrodynamic diameter (Dh) of copolymers and glycopolymers were measured by
dissolving the sample in water (5 mg mL−1) and filtering through a 0.45 µm PVDF filter.
Nano-ZS instrument (ZEN3600, Malvern, UK) was used and the intensity of the scattered
light was detected at angle θ = 173° using laser of a wavelength of 632.8 nm. The values
were determined as a mean of at least three independent measurements.
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UV–VIS Spectrophotometry
The content of thiazolidine-2-thione (TT) groups in the copolymer precursors were determined
by UV–VIS spectrophotometry in methanol using Specord 205 ST, Analytic Jena AG, Germany.
The molar absorption coefficient used was ε(TT) = 10,300 L mol−1 cm−1 (λmax = 305 nm)
estimated for 3,3′-disulfanediylbis[1-(2-thioxothiazolidin-3-yl)propan-1-one].
Enzyme Activity Assay
The activity of the β-galactosidase of Bacillus circulans in the commercial preparation Biolacta
BN5 (Daiwa Kasei, Ltd) was measured in an end-point assay with p-nitrophenyl β-D-
galactopyranoside (1; pNP-Gal; Glycon Biochemicals, Germany) as a substrate at a starting
concentration of 2 mM. The reaction mixture was incubated in 50 mM sodium acetate buffer pH
5 for 10 min at 35 °C and 850 rpm. Then, the reaction (50 µL) was stopped by adding 0.1 M
Na2CO3 (1 mL) and the amount of released p-nitrophenol was determined
spectrophotometrically at 420 nm. One unit of enzymatic activity corresponds to the amount of
enzyme releasing 1 µmol of p-nitrophenol per minute under the above conditions. The
commercial preparation contained a side activity of β-N-acetylhexosaminidase, which was
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determined analogously using p-nitrophenyl 2-acetamido-2-deoxy-β-D-glucopyranoside as a
substrate (pNP-GlcNAc; Sigma-Aldrich, USA). In reactions containing co-solvents, the
respective amount of buffer was substituted by the respective co-solvent (acetone or acetonitrile)
in 0-40% v/v, and the enzyme activity was determined as described above. All data were
measured in triplicate.
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Synthetic Procedures
2-Acetamido-2-deoxy-β-D-glucopyranosyl
azide
(2)
and
(t-
butoxycarbonylamido)ethylthioureidyl 2-acetamido-2-deoxy-β-D-glucopyranoside (3)
Compound 233 and compound 334 were prepared from 2-acetamido-2-deoxy-D-glucopyranose
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(GlcNAc; Acros Organics) using an optimized procedure described previously. The H and C
NMR data corresponded to the structure.
Optimization of Enzymatic Synthesis of Disaccharides 4 and 5 at Analytical Scale
pNP-Gal donor (1; 30-50 mM) was combined with 2-acetamido-2-deoxy-β-D-glucopyranosyl
azide acceptor (2; 150–250 mM) or with (t-butoxycarbonylamido)ethylthioureidyl 2-acetamido-
2-deoxy-β-D-glucopyranoside acceptor (3; 150–250 mM). The mixtures were dissolved in 50
mM sodium acetate buffer (pH 5.0-7.5) with the addition of acetonitrile (0-40 % v/v). The β-
galactosidase from B. circulans (0.03–0.16 mg mL−1, 0.05–0.3 U mL−1) was added and the
reaction mixtures were shaken at 35 °C and 850 rpm for up to 36 h. Aliquots (10 μL) were
regularly analyzed by TLC (propan-2-ol/H2O/NH4OH, 7/2/1, v/v/v) with detection under UV
light and by carbonization, and by HPLC.
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β-D-Galactopyranosyl-(14)-2-acetamido-2-deoxy-β-D-glucopyranosyl azide (4)
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Compound pNP-Gal (1; 50 mg, 0.17 mmol), acceptor 2 (185 mg, 0.75 mmol) and the β-
galactosidase from B. circulans (0.4 U, 0.75 mg, 15 μL) were incubated in a mixture of
acetonitrile (20 % v/v) in 50 mM sodium acetate buffer pH 5.0 (total reaction volume 5 mL) at
35 °C and 850 rpm under monitoring by HPLC and TLC. After 24 h, another portion of enzyme
(0.04 U, 0.38 mg, 8 μL) was added and the reaction ran for additional 4 h. Then the reaction
mixture was boiled for 2 min to inactivate the enzyme, cooled down to room temperature (r.t.),
and centrifuged (13,500 rpm; 10 min). For product isolation, it was loaded onto a Biogel P-2
column (26 × 1000 mm, Bio-Rad, USA) with water mobile phase at 7 mL h-1. Pure acceptor 2
was partially recovered (115 mg). The fractions containing the product were combined and
lyophilized; the title compound 4 was obtained as a white solid (29 mg, 0.07 mmol, 56 %
isolated yield). HRMS (ESI−): found m/z 407.14148 (expected 407.14197 for [M−H]−,
C14H22O10N4-), Fig. S5 in the ESI. For the respective NMR data, see the ESI: Table S1 and
Figures S1a and S1b.
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(t-Butoxycarbonylamido)ethylthioureidyl
β-D-galactopyranosyl-(14)-2-acetamido-2-
deoxy-β-D-glucopyranoside (5) and (2-aminoethyl)thioureidyl β-D-galactopyranosyl-(14)-
β-D-glucopyranoside (6)
Compound pNP-Gal (1; 50 mg, 0.17 mmol), acceptor 3 (318 mg, 0.75 mmol) and the β-
galactosidase from B. circulans (0.3 U, 0.56 mg, 11 μL) were incubated in 50 mM sodium
acetate buffer pH 5.0 (total reaction volume 5 mL) at 35 °C and 850 rpm under monitoring by
HPLC and TLC. After 5 h, the reaction was boiled for 2 min to inactivate the enzyme, cooled
down to r.t., and centrifuged (13,500 rpm; 10 min). For product isolation, it was loaded onto a
Biogel P-2 column with water mobile phase at 7 mL h-1. Pure acceptor 3 was partially recovered
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(195 mg). The fractions containing the product were combined and lyophilized; the title
compound 5 was obtained as a white solid (31 mg, 0.05 mmol, 31 % isolated yield). HRMS
(ESI−): found m/z 583.22814 (expected 583.22907 for [M−H]−, C22H39O12N4S-), Fig. S6 in the
ESI. For the respective NMR data, see the ESI: Table 3 and Figures S3a and S3b.
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For conjugation to the polymer carrier, the t-butoxycarbonyl (t-Boc) protecting group was
deprotected by dissolving disaccharide 5 (10 mM) in 1M HCl and incubating under stirring at 4
°C for 48 h. Then, neutralization by Amberlite anex resin (OH− cycle) was performed and the
deprotected compound 6 (16 mg, 0.03 mmol, 64 % yield) was lyophilized. HRMS (ESI−): found
m/z 483.17664 (expected 483.17615 for [M−H]−, C17H31O10N4S-), Fig. S7 in the ESI. The
deprotected amine 6 (see the ESI: Table 4, Figs. S4a and S4b) should be readily used for
conjugation as it tends to decompose under long-term storage.
Synthesis of Branched Linkers 14 and 15
The branched linkers containing either two triple bonds (14) or three triple bonds (15)
were prepared in three steps. In the first step, compounds 10 (470 mg, 65 %) and 11 (181
mg, 56 %) containing alkyne were prepared from methyl 3,5-dihydroxybenzoate (8) or
methyl-3,4,5-trihydroxybenzoate (9), respectively, using propargyl bromide and K2CO3 in
acetone, as previously described.35 Then, intermediates 10 and 11 reacted with KOH in
ethanol/water according to Deng et al.,36 yielding intermediates 12 (184 mg, 83 %) and 13
(113 mg, 65 %), respectively. The title branched linkers containing t-Boc group
protecting primary amino group, namely compounds 14 (198 mg, 67 %) and 15 (82 mg,
64%) were obtained by amide formation of compounds 12 and 13, respectively, using N-
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Boc-ethylenediamine via a modified procedure previously described.37 The detailed
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6
synthetic procedures, NMR and MS data are shown in the ESI, Section 3.1.
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Synthesis of Glycosylated Branched Linkers 18 and 19
Glycosylated branched linkers 18 and 19 were synthesized in two steps. First, branched
linkers 14 or 15 reacted with disaccharide 4 by Cu(I)-catalyzed azide-alkyne
cycloaddition (CuAAC) in DMF overnight, affording compounds 16 and 17, respectively.
Then, the t-Boc protecting group was removed under acidic conditions in CH2Cl2 for 2 h,
yielding primary amines (18 and 19) to be coupled to polymer precursors.
The detailed synthetic procedure for compound 18 is as follows: Compounds 14 (15 mg,
40 µmol) and 4 (37.8 mg, 93 µmol) were dissolved in 1 mL of dry DMF and the mixture
was bubbled with argon for 30 min. Then, CuBr (3.5 mg, 24 µmol; powder form) was
added, and the reaction mixture was kept under argon for 1.5 h. Then, after changing the
septum, the reaction was carried out under argon atmosphere and stirring at r.t. for 20 h.
DMF was removed under vacuum by repeated co-distillation with methanol and water
and purification was performed using gel permeation chromatography on Biogel P-2
column with water as a mobile phase (7 mL h-1), yielding compound 16 (40 mg, 83%).
For NMR data, see Table S5 and Figs. S11a and S11b in the ESI, Section 3.2. For MS
data, see Fig. S11c in the ESI, Section 3.2. For t-Boc deprotection, compound 16 (40 mg,
33.6 µmol) was mixed with CH2Cl2 (4 mL), then TFA (342 µL, 4.5 mmol) was added
dropwise in three steps (1.5 mmol in each step) with 30 min intervals under cooling and
stirring. The reaction was monitored by HPLC and TLC (propan-2-ol/H2O/NH4OH,
7/2/1, detection by carbonization using 5% v/v H2SO4 in ethanol). After shaking at r.t. for
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2 h, CH2Cl2 was evaporated under vacuum. TFA was removed by repeated co-distillation
with methanol and water under vacuum until neutral pH was reached. Evaporation under
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1
vacuum yielded the title compound 18 (34 mg, 85%). H NMR (600.2 MHz, D2O): δ ≈
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8.28 ppm (s, 2H, CH from triazol of both meta positions), δ ≈ 7.04 ppm (s, 2H, CH from
ortho positions of aromatic portion), δ≈ 6.84 ppm (s, 1H, CH para position of aromatic
portion), δ ≈ 5.85 ppm (d, 2H, CH from C-1 of GlcNAc units), δ ≈ 5.26 ppm (s, 4H, CH2
next to triazol of meta positions), δ ≈ 4.52 ppm (d, 2H, CH from C-1 of Gal units), δ ≈
4.30 ppm (t, 2H, CH of C-2 of GlcNAc units), δ ≈ 3.93 ppm (m, 12H, CH of C-3 and C-4
of GlcNAc units, CH2 of C-6 of GlcNAc units, CH of C-4 of Gal units and 1H, CH2 of C-
6 of Gal units), δ ≈ 3.75 ppm (m, 6H, CH of C-5 of GlcNAc units, CH of C-5 of Gal units
and 1H, CH2 of C-6 of Gal units), δ ≈ 3.68 ppm (m, 4H, CH of C-2 of Gal units and 2H,
CH2–NH), δ ≈ 3.57 ppm (t, 2H, CH of C-3 of Gal units), δ ≈ 3.25 ppm (t, 2H, CH2–NH2),
δ ≈ 1.67 ppm (s, 6H, CH3CONH~ of GlcNAc units). See also Fig. S12 in the ESI, Section
3.2.
The synthetic procedure for compound 19 was analogous to 18. The reaction of
compounds 15 (11 mg, 25 µmol) and 4 (33 mg, 81 µmol), using CuBr catalysis (3.5 mg,
24 µmol; powder form) in dry DMF (1.2 mL) afforded compound 17 (29 mg, 66%). For
NMR data, see Table S6 and Figs. S13a and S13b in the ESI, Section 3.2. For MS data,
see Fig. S13c in the ESI, Section 3.2. For t-Boc deprotection, compound 17 (29 mg, 17.5
µmol) reacted with TFA (170 µL, 2.2 mmol) in CH2Cl2 (4 mL), yielding the title
compound 19 (28 mg, 98%). 1H NMR (600.2 MHz, D2O): δ ≈ 8.26 ppm (s, 2H, CH from
triazol of both meta positions), δ ≈ 8.14 ppm (s, 1H, CH from triazol of para position), δ
≈ 7.20 ppm (s, 2H, CH from ortho positions of aromatic portion), δ ≈ 5.86 ppm (d, 2H,
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CH from C-1 of GlcNAc units from meta positions), δ ≈ 5.80 ppm (d, 1H, CH from C-1
of GlcNAc moiety from para position), δ ≈ 5.25 ppm (br, 4H, CH2 next to triazol of meta
positions), δ ≈ 5.16 ppm (br, 2H, CH2 next to triazol of para position), δ ≈ 4.53 ppm (d,
3H, CH from -1 of Gal units), δ ≈ 4.30 ppm (m, 3H, CH from C-2 of GlcNAc units), δ ≈
3.93 ppm (m, 18H, CH from C-3 and C-4 of GlcNAc units, CH2 from C-6 of GlcNAc
units, CH of C-4 of Gal units and 1H, CH2 of C-6 of Gal units), δ ≈ 3.76 ppm (m, 9H, CH
of C-5 of GlcNAc units, CH of C-5 of Gal units and 1H, CH2 of C-6 of Gal units), δ ≈
3.68 ppm (m, 5H, CH of C-2 of Gal units and 2H, CH2–NH), δ ≈ 3.57 ppm (t, 3H, CH of
C-3 of Gal units), δ ≈ 3.25 ppm (t, 2H, CH2–NH2), δ ≈ 1.75 ppm (s, 3H, CH3CONH~ of
GlcNAc unit of para position), δ ≈ 1.65 ppm (s, 6H, CH3CONH~ of GlcNAc units of
meta positions). See also Fig. S14 in the ESI, Section 3.2.
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Synthesis of Copolymer Precursors 22-24
The monomers HPMA (20) and MA-AP-TT (21) were synthesized as described
previously.38,39,40 The poly(HPMA-co-MA-AP-TT) polymer precursors 22-24 were
prepared by RAFT polymerization using CTA and the initiator AIBN, followed by
dithiobenzoate end group removal as shown before.41 The reaction conditions were
adopted from our previous work40 and the molar ratio of monomer/CTA/AIBN was
350/2/1. The molar ratio of monomers HPMA/MA-AP-TT varied as follows: 90/10
(copolymer 22; 9 mol. % of TT groups), 80/20 (copolymer 23; 17 mol. % of TT groups)
or 75/25 (copolymer 24; 23 mol. % of TT groups). The detailed synthetic procedures for
copolymers 22-24 are described in the ESI, Section 4.1.
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Synthesis of Glycopolymers with Individual LacNAc Presentation (25-29)
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8
The glycopolymers 25-29 with individual presentation of LacNAc were prepared by
aminolysis of TT groups of polymer precursors 22-24 with the corresponding amount of
disaccharide 6. Glycopolymers 25 and 26 were prepared from polymer precursor 22,
glycopolymers 27 and 28 from precursor 23 and glycopolymer 29 from precursor 24. A
representative synthetic procedure for glycopolymer 28 is as follows. Disaccharide 6 (12
mg, 22 µmol) was dissolved in a mixture of dry methanol and dry DMA (1/3; 800 µL)
and added dropwise to the solution of copolymer precursor 23 (20 mg; 27 µmol of TT
groups) in dry methanol (200 µL). After stirring for 30 min, DIPEA was added (4.9 µL,
28 µmol) and the reaction was carried out under stirring for 20 h at 24 °C. Then, 1-
aminopropan-2-ol (2 µl, 27 µmol) was added, and the reaction was left stirring for 30 min
to remove the unreacted TT groups. Crude glycopolymer 28 was diluted with methanol
(1.2 mL), and purified on Sephadex LH-20 column in methanol mobile phase under UV
detection. The combined polymer fractions were evaporated under vacuum, dissolved in
distilled water and freeze-dried (26 mg, 88 %). NMR data for glycopolymer 28 are shown
in the ESI, Section 4.2., Fig. S15. For the calculation of LacNAc content by NMR
analysis, the integral intensity of signal at δ = 3.92 ppm (1H, CH–OH) or δ = 3.00-3.28
ppm (2H, CH2–NH) of the HPMA unit, and of signal at δ ≈ 4.48 ppm (1H, CH of C-1 of
Gal) of LacNAc were used. Molecular weights and dispersities of glycopolymers 25-29
are shown in Table 1.
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Synthesis of Glycopolymers with Clustered LacNAc Presentation: Bivalent (30-34) and
Trivalent (35-39) Branching
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The glycopolymers 30-34 carrying bivalent branching and the glycopolymers 35-39
carrying trivalent branching were prepared by aminolysis of the TT groups of the polymer
precursor 23 with the primary amino group of compounds 18 and 19, respectively. A
representative synthetic procedure for glycopolymer 36 with trivalent branching is as
follows. Copolymer 23 (24 mg; 25 µmol of TT groups) was dissolved in dry DMA (200
µL) and compound 19 (6.0 mg, 3.6 µmol) dissolved in dry DMA/methanol (3/1; 1 mL)
was added. After stirring for 1 h, DIPEA (0.9 µL, 5 µmol) was added, and the reaction
was carried out at 24 °C for 20 h. To remove the unreacted TT groups, of 1-aminopropan-
2-ol (1.8 µL, 24 µmol) was added and the reaction mixture was stirred for 30 min. Crude
glycopolymer 36 was purified on Sephadex LH-20 column with methanol mobile phase
under UV detection. The collected polymer fraction was evaporated under vacuum,
dissolved in water and freeze-dried (25 mg, 86 %). NMR data for glycopolymers 31 and
38 are shown in the ESI, Section 4.2., Figs. S16 and S17, respectively. For the calculation
of disaccharide content by NMR analysis, the integral intensity of signal at δ = 3.00-3.28
ppm (2H, CH2–NH) of the HPMA unit were used. The LacNAc content in glycopolymers
30-34 was calculated using signals at δ ≈ 8.31 ppm (2H, CH of both triazol moieties), δ ≈
5.86 ppm (2H, CH of C-1 of GlcNAc), δ ≈ 4.51 ppm (2H, CH of C-1 of Gal), δ ≈ 4.30
ppm (2H, CH of C-2 of GlcNAc). The LacNAc content in glycopolymers 35-39 was
calculated using signals at δ ≈ 8.30 ppm (2H, CH of triazol of both meta positions), δ ≈
8.14 ppm (1H, CH of triazol of para position), δ ≈ 7.20 ppm (2H, CH of ortho position of
aromatic portion), δ ≈ 5.90 ppm (2H, CH of C-1 of GlcNAc in meta positions), δ ≈ 5.81
ppm (1H, CH of C-1 of GlcNAc in para position), δ ≈ 4.32 ppm (3H, CH of C-2 of
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GlcNAc). Molecular weights and dispersities of glycopolymers 30-39 are shown in Table
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6
1.
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Protein Production
Recombinant Human His6-tagged Galectin-1 and Galectin-3
Recombinant His-tagged constructs of Gal-1 and Gal-3 were produced and purified as
described previously.8 Gal-1 construct contained Cys2Ser mutation for increasing its
stability.42,43 Briefly, both galectins were recombinantly expressed in the cells of E. coli
Rosetta 2 (DE3) pLysS. The cultivation of pre-cultures (60 mL in 0.5 L baffled flasks)
was done overnight in Lysogeny broth (LB) medium (0.5 w/v yeast extract, 1.0 v/w
tryptone, 0.5% w/v NaCl pH 7.4) at 220 rpm and 37 °C. The medium contained
ampicillin (100 mg L-1) and chloramphenicol (34 mg L-1). After 17 h, main cultures (600
mL in 3L baffled flasks) in Terrific broth (TB) medium (2.4 w/v yeast extract, 1.2% w/v
tryptone, 0.4% v/v glycerin, 17 mM KH2PO4, 72 mM K2HPO4 pH 7.0) containing
antibiotics were inoculated with pre-cultures and incubated at 37 °C and 150 rpm until
they reached optical density (OD600) of 0.6–0.8. Then, the expression of galectins was
induced by isopropyl 1-thio-β-D-galactopyranoside (0.5 mM). After 24 h, the cells were
centrifuged (5,000 rpm, 20 min, 4 °C) and frozen at -20 °C. For purification of galectins,
E. coli cells were suspended in ice-cold equilibration buffer (20 mM Na2HPO4, 500 mM
NaCl, 20 mM imidazole, pH 7.4) and sonicated on ice (six 30 s cycles, 52% amplitude).
After removing cell debris (centrifugation at 13,400 rpm, 15 min, 10 °C), the supernatant
was filtered through 0.8 µm syringe filter and loaded on HisTrap™ HP 5 mL column (GE
Healthcare) according to manufacturer`s instructions in equilibration buffer. Galectins
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were eluted by elution buffer (20 mM Na2HPO4, 500 mM NaCl, 500 mM imidazole, pH
7.4). The combined fractions containing galectin were dialyzed in SnakeSkin™ Dialysis
Tubing (10 kDa MWCO, ThermoFisher Scientific) overnight against 50 mM
NaH2PO4/150 mM NaCl/2 mM ethylenediaminetetraacetic acid pH 7.5 (EPBS buffer),
and for additional 4 h against 50 mM NaH2PO4/150 mM NaCl pH 7.5 (PBS buffer). A
usual yield was ca 7-10 g of cells per 1 L medium and 5 mg of pure Gal-3 and 15 mg of
pure Gal-1 per 1 g cells. After sterile filtration, galectins (100-200 μM solutions) were
stable for 1 - 2 months; due to lower stability, galectin-1 solution contained glycerol (20
% v/v). The galectin samples contained ca 9000-10,000 E.U. (i.e. 900-1000 ng)
lipopolysaccharide (LPS) per mg protein as determined by chromogenic Endpoint
Chromogenic Limulus Amebocyte Lyzate (LAL) Kit (Charles River Endosafe,
Charleston, USA). These LPS concentrations were confirmed to have no influence on the
outcome of ELISA-type measurements. Alternatively, LPS-free purification of galectins
was performed by modifying the standard procedure: an additional step of washing the
loaded sample with 0.5% v/v Triton X-100 in equilibration buffer (10 column volumes),
followed by extensive washing with equilibration buffer (20 column volumes) was
included, reducing LPS content ca 20-times. This optional step had no impact on galectin
viability or biological activity.
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Competitive ELISA Assay with Galectin-1 and Galectin-3
The ability of glycopolymers 25-39 to inhibit binding of soluble human Gal-1 or Gal-3 to
immobilized standard ligand asialofetuin (ASF, Sigma Aldrich, Steinheim, Germany) was
determined in a competitive ELISA assay. For reference, binding affinities of both
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galectins to immobilized ASF (Kd,app) were determined in a direct ELISA assay,19,44 and
were in correlation with previously published results44 acquired by the same method:
Kd,app = (2.6  0.6) M for Gal-1 vs. (3.7  0.5) M for Gal-3. The F16 Maxisorp NUNC-
Immuno Modules (Thermo Scientific, Roskilde, Denmark) were coated with ASF (0.1
μM, 50 μL/well) in PBS buffer (50 mM NaH2PO4/150 mM NaCl pH 7.5) overnight.
Then, blocking with BSA (2% w/v) in PBS followed (1 h, r.t.). Afterwards, respective
glycopolymers in various concentrations and Gal-1 or Gal-3 (total volume 50 μL; 4.5 μM
final galectin concentration) in EPBS buffer were added, and incubated for 2 h. After
each step, the wells were extensively rinsed with Tween 20 (0.05% v/v) in PBS. Bound
galectins were detected using anti-His6-IgG1 mouse antibody conjugated to horseradish
peroxidase (Roche Diagnostics, Mannheim, Germany) in PBS (1/500 for Gal-1 and
1/1000 for Gal-3, 1 h, r.t., 50 μL/well). The IgG-conjugated peroxidase was quantified by
TMB One substrate (Kem-En-Tec, Taastrup, Denmark). The reaction was stopped by
adding 3M HCl (50 μL) for colorimetric detection at 450 nm. LacNAc (7) was used as a
positive control.
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RESULTS AND DISCUSSION
Chemoenzymatic Synthesis of Functionalized Disaccharides 4 and 5
In order to compare two different presentations of LacNAc (7; Galβ4GlcNAc; for
structure see Scheme 2) epitope on the HPMA copolymer backbone, we prepared
LacNAc disaccharide with two different functionalities by enzymatic synthesis.
Glycosidases are robust glycosylation tools tolerating a variety of modifications in the
substrate molecule,45,46 although they are unable to catalyze multifold glycosylation of a
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multivalent acceptor.47 The amino function in disaccharide 6 was used, after t-Boc
deprotection, for direct coupling to HPMA polymer precursors 22-24 by aminolysis
whereas the azido function in disaccharide 4 enabled nested presentation on a bi- or
trivalent aromatic linkers (14 and 15, respectively) using CuAAC. Both functionalized
disaccharides 4 and 6 were prepared from respective monosaccharide precursors 3 and 2
by one-step synthesis in aqueous buffer using a commercial preparation Biolacta® with
β-galactosidase from Bacillus circulans. This enzyme is well known as a robust and
potent galactosylation tool used especially for industrial preparation of long-chain
galactooligosaccharides.48,49 In this case, our task was different since we aimed at a
regioselective and high-yielding mono-galactosylation of two different functionalized
acceptors. Both acceptors 2 and 3 were prepared as described previously.33,34 The t-Boc
protected compound 3 proved to be an attractive acceptor for β-galactosylation, yielding
the desired disaccharide 5 in a yield of 31 %. After t-Boc deprotection, the resulting
compound 6 was readily coupled to respective polymer precursors.
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NO2
OH
2
-O
OH
O
OH
O
OH
O
6
7
8
9
HO
HO
N3
N3
O
HO
HO
NH
NH
OH
O
O
OH
i
OH
10
11
12
13
14
15
16
17
18
19
20
21
22
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4
O
O
-galactosidase
OH
O
OH
6
HO
OH
OH
from Bacillus circulans
H
N
H
O
1
N
NO2
O
HO
HO
NH2
NH
OH
S
ii
O
OH
iii
3
O
OH
H
H
OH
O
OH
HO
HO
N
N
O
H
H
O
N
O
O
N
N
O
NH
H
S
HO
HO
-O
N
O
O
H
NH
OH
S
O
NO2
5
Scheme 1. Enzymatic synthesis of functionalized LacNAc epitope. i, 20% CH3CN in 50 mM
sodium acetate buffer pH 5.0, 28 h, 35 °C; ii, 50 mM sodium acetate buffer pH 5.0, 5 h, 35 °C;
iii, 1M HCl, 4 °C, 48 h.
The synthesis of compound 4 was much more challenging. Besides β-galactosidase, the
Biolacta preparation also contains contaminating β-N-acetylhexosaminidase. The latter
activity is known to cleave acceptor 2,33 which considerably lowered product yields.
Moreover, depending on its set-up, the transgalactosylation was not fully regioselective
since the Galβ6GlcNAc azide regioisomer was sometimes formed (see the ESI, Table S2
and Figs. S2a and S2b). Therefore, we applied extensive reaction engineering in order to
increase transgalactosylation yields and render the reaction fully regioselective. The
dependence of β-galactosidase and β-N-acetylhexosaminidase activities on reaction pH
(see the ESI, Fig. S8A) indicated pH  5 or ≥ 7 as the most selective for β-galactosidase
activity. Analysis of transgalactosylation reactions at various pH revealed that the
reaction regioselectivity strongly depends on pH; whereas at slightly basic pH a mixture
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of β4 (compound 4) and β6 regioisomers were formed in a ratio of ca 3/1, the reaction at
pH 5 proved to be fully regioselective yielding exclusively compound 4. Since at pH 5 the
residual β-N-acetylhexosaminidase activity still lowered the synthetic yield, we screened
various co-solvents for its further suppression (ESI, Fig. S8B, C). Whereas increasing
concentrations of acetone (0-40% v/v) suppressed both activities rather equally,
acetonitrile discriminated β-N-acetylhexosaminidase faster than β-galactosidase. Thus, in
20 % v/v acetonitrile, residual β-N-acetylhexosaminidase activity was only 6% compared
to 60% maintained β-galactosidase activity. Additionally, acetonitrile has a higher boiling
point than acetone, and it had already been found useful in similar reactions. Therefore,
we opted for a final setup of 20% v/v acetonitrile/acetate buffer pH 5.0. It afforded the
desired product 4 in 56% yield, to be coupled to branched linkers 14 and 15.
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14
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Synthesis of Glycosylated Branched Linkers 18 and 19
Bi- and trivalent branched linkers 14 and 15, respectively, (Scheme 2A) were prepared by
an original procedure in three steps. First, intermediates 10 and 11 were afforded by
propargylation of compounds 8 and 9, respectively. Complete propargylation was
1
confirmed by the appearance of respective alkyne signals in H NMR spectrum.
Subsequent methyl ester hydrolysis yielded intermediates 12 and 13, and we observed the
1
disappearance of methyl ester signal in H NMR spectrum. The final step, amide
formation yielding 14 and 15, was accompanied by appearance of signals of amide,
1
urethane and t-Boc groups in H NMR spectrum. Azido-functionalized LacNAc (4) was
conjugated to the available propargyl moieties on 14 or 15 by CuAAC (one set-up of
1
“click chemistry”),50 accompanied by the appearance of the triazole moiety signal in H
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NMR spectrum. The subsequent t-Boc deprotection afforded the title compounds 18 and
19 with two or three LacNAc moieties, respectively, and an amino group for the
attachment to polymer precursors. The t-Boc deprotection proved to be the most
challenging part of the synthesis. Here, we had to optimize the method to prevent the
cleavage of the glycosidic bond and achieve the complete deprotection. Finally, we found
that a reasonable approach to tackle this issue is to perform the reaction by a careful
addition of a total of 132 equivalents of TFA in three steps (44 eq. added dropwise in
each step) under cooling, and terminate the reaction after a maximum of 2 h under
monitoring by HPLC.
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Synthesis and characterization of polymer precursors 22-24 and glycopolymers 25-
39
Polymer precursors 22-24 containing reactive TT groups with comparable molecular
weights (Mn ≈ 19,000 - 24,600 g mol-1) and narrow dispersity (Đ ≈ 1.1) were prepared by
controlled RAFT polymerization technique. The copolymers differed in the content of the
TT reactive groups, i.e., 9 mol. % in 22, 17 mol. % in 23 and 23 mol. % in 24. The
varying content of TT groups enabled the covalent attachment of the amine-
functionalized LacNAc (6) or amine-terminated branched linkers bearing two or three
LacNAc glycans (18 and 19, respectively) in a wide content range (see Table 1).
The aminolysis of TT groups by disaccharide 6 afforded glycopolymers 25-29 containing
ca 5 to 19 mol. % LacNAc statistically distributed along the polymer chain (Scheme 2B).
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A
8, 9
10, 11
12, 13
Br
R2
R1
R2
KOH
O
O
HO
OH
O
O
7
8
9
acetone, K2CO3,
70 oC, 20 h reflux
EtOH/H2O (9/1), r.t.,
18 h, pH adjustment
HO
O
O
O
O
O
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
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ethylen
PyBOP,
N
-Boc
eadiami
DCM,
r.t.,
DIPEA,
16
ne
h
16, 17
14, 15
18, 19
R3
R3
R3
R3
4
R2
R4
R4
N
N
N
N
N
N
N
N
N
N
O
O
O
O
O
O
N
N
Cu(I)
DMF, r.t., 20 h
TFA, CH2Cl2, 2 h,
pH adjustment
HN
O
HN
O
HN
O
O
NH
NH3+ TFA-
O
NH
O
O
B
S
S
20
21
22 24
25 29
-
-
6
N
/
/
/
O
O
O
O
O
O
O
O
HN
HO
HN
HN
HN
HN
HN
HO
+
HN
HN
HO
HN
1. DIPEA, DMA/MeOH, r.t., 20 h
2. 1-aminopropan-2-ol, 30 min
1. AIBN, t-BuOH/DMA,
70 oC, 16 h
2. AIBN, DMA, 80 oC, 3 h
O
O
N
O
S
N
S
S
HO
S
NH
NH
S
1.
2. DIPEA,
1-amin
R2
R1
=
18, 19
=
R3
10, 12, 14
8
(
)
(
)
H
DMA/MeOH,
opropan
H
30 39
-
9
(
)
11, 13, 15
(
)
-2-ol,
O
HO
r.t.,
30
/
/
20
min
h
O
O
O
R4
HN
HN
HN
HN
R3
=
HN
HO
=
16, 18, 30 34
)
(
-
H
OH
O
OH
O
OH
HO
O
O
N
17, 19, 35 39
)
(
-
O
O
N
N
HO
O
NH
OH
HO
NH
R3
O
N
N
N
OH
O
OH
O
OH
HO
O
R3
O
OH
O
HO
R4
NH
OH
N
O
N
N
LacNAc (Gal4GlcNAc)
R3
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Scheme 2 (A), Synthesis of glycosylated branched linkers 18 and 19; (B), synthesis of
glycopolymers 25-29 with individual presentation of LacNAc, and glycopolymers 30-39
with LacNAc presented on bi- and trivalent branched linkers. Structure of LacNAc
disaccharide is shown in the box.
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11
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The molar mass and size of the polymers increased with the increasing amount of
LacNAc content, showing the slight expansion of the polymer coil. Only in
glycopolymers 28 and 29 with a high LacNAc content, the dispersity slightly increased.
We assumed that this increase could be ascribed to the statistical distribution of
disaccharide along the polymer backbone since any slight difference in the disaccharide
content between various polymer chains will be more notable at higher LacNAc contents.
Moreover, we also observed limited solubility of 28 and 29 in methanol, which might
have been caused by the high amount of LacNAc. Therefore, the molecular weight of 28
and 29 was determined in aqueous mobile phase on the Superose column.
The glycopolymers carrying LacNAc epitope presented on dibranched (30-34) or
tribranched (35-39) linkers were prepared by conjugation of primary amino group of the
glycosylated compounds 18 or 19, respectively, with the reactive TT groups of polymer
precursor 23 (Scheme 2B). The content of LacNAc ranged from ca 4 to 22 mol. %, and
the introduction of branched linkers containing LacNAc in bi- or trivalent presentation led
to a slight increase in the polymer molar mass. Similar to glycopolymers with
disaccharide 6, a slight increase in the hydrodynamic size of glycopolymers 30-39 was
observed with increasing amount of di- or tribranched LacNAc epitopes. In
glycopolymers 33, 34, 38, and 39 with a high content of LacNAc we also observed a
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